The present invention relates to plant molecular biology, to signal transduction from outside the plant to the nucleus and to systems for sensing a target substance of interest in the environment and inducing gene expression in response thereto, along with a novel plant biomarker useful for reporting the detection of biological and chemical agents and environmental pollutants based on the loss of green color in plants. All publications cited in this application are herein incorporated by reference.
There have been increasing threats from terrorists which present an urgent need for simple and robust detectors for harmful biological or chemical agents. Current detectors of biological and chemical agents involve electronic and/or vacuum-like mechanisms to sample the air or the environment. All current means to detect terrorist agents are costly and require continuous maintenance. The high and continuous cost significantly limits the ability to detect biological and chemical weapons as well as environmental pollutants.
Each fall, plants display a dramatic loss in green color. The bright yellow, red, and orange colors are unmasked by the tremendous loss in the green chlorophyll pigments. Consequently, the loss of green color in plants is a phenomenon that most people readily recognize. This metabolic degreening process is not unique to the foliage of deciduous trees. A metabolic degreening is found in all plants including evergreens and algae (Matile et al., 1999).
The green color in plants is due to a pigment known as chlorophyll. Most higher green plants contain two types of chlorophyll, chlorophyll a and chlorophyll b. Each molecule has a porphyrin-like ring attached to a long hydrocarbon tail. Chlorophyll a and chlorophyll b differ only in a side group of ring II. In most plants, chlorophyll a is the dominant form with lesser amounts of chlorophyll b. The two forms of chlorophyll undergo a simple cyclic interconversion. Chlorophyll is synthesized in the a form and can be converted to the b form through chlorophyll a oxygenase; chlorophyll b is converted back to chlorophyll a through the action of chlorophyll b reductase (Malkin and Niyogi, 2000; Thomas et al., 2002).
The biosynthetic pathway for chlorophyll is very well known (Malkin and Niyogi, 2000). Chlorophyll biosynthesis begins with glutamic acid. Through nine biochemical steps glutamic acid is converted to a four-ring structure, protoporphyrin IX. Magnesium chelatase adds magnesium to the ring structure. In two additional steps, monovinyl protochlorophyllide a is formed. The enzyme protochlorophyllide oxidoreductase (POR) reduces the monovinyl protochlorophyllide molecule to form chlorophyllide a. Importantly, the POR enzyme controls the rate-limiting step in chlorophyll biosynthesis. Chlorophyllide a has a light green color and differs from chlorophyll by lacking the long hydrocarbon tail. The chlorophyllide molecule is converted to the darker green chlorophyll molecule by the enzyme chlorophyll synthetase, which adds a twenty-carbon phytol tail. Like most biological molecules, steady state levels of chlorophyll are maintained by a combination of biosynthesis and catabolism with the half-life of chlorophyll in a green plant being approximately 50 hours (Matile et al., 1999).
Like chlorophyll biosynthesis, the chlorophyll breakdown pathway is also very well characterized (Matile et al., 1999; Tsuchiya et al., 1999; Dangl et al., 2000). Chlorophyllase, one of the major enzymes involved in the first step of chlorophyll breakdown, removes the hydrophobic, twenty carbon phytol tail from chlorophyll (Matile et al., 1999; Tsuchiya et al., 1999; Dangl et al., 2000; Benedetti and Arruda, 2002). Similar to the biosynthetic pathway, chlorophyll without the phytol tail becomes the light green molecule, chlorophyllide. The lack of the phytol tail also changes solubility; chlorophyllide is soluble in aqueous solutions whereas chlorophyll is soluble in organic solvents.
The chlorophyllide a molecule is converted to pheophorbide a by removal of the magnesium by the enzyme magnesium dechelatase (Matile et al., 1999; Dangl et al., 2000; Takamiya et al., 2000). A red-colored compound, red chlorophyll catabolite (RCC), forms next through the action of the enzyme pheophorbide a oxygenase (Hortensteiner et al., 1998; Thomas et al., 2002). Next, the enzyme RCC reductase acts to produce fluorescent chlorophyll catabolite (FCC). Subsequently, various enzymes convert FCC to nonfluorescent chlorophyll catabolites. Nonfluorescent chlorophyll catabolite molecules accumulate in the plant's vacuole.
Importantly, the chlorophyll degradation pathway is not thought to be part of the system involved in steady-state regulation of chlorophyll levels, because chlorophyll catabolites have never been found in green cells with steady chlorophyll levels (Matile et al., 1999). This suggests that induction of genes in the chlorophyll degradation pathway will lead to the rapid breakdown of chlorophyll. Indeed, transgenic plants with constitutive expression of the chlorophyllase gene had massive accumulation of the enzyme's product, chlorophyllide (Benedetti and Arruda, 2002). These plants retained the ability to synthesize chlorophyll and hence retained a green color.
Plants, because of their sessile nature, have evolved sophisticated mechanisms for sensing and responding to their environment and substances in their environment (Trewavas, 2000, 2002). The presence of a plant cell wall, now understood to be a complex matrix, does not deter the ability of green plants to detect analytes (Dangl et al., 2000). Indeed, plants are capable of detecting analytes intracellularly (e.g., soluble or cytoplasmic analytes such as chemicals or phytohormones) or extracellularly (e.g., certain pathogens, chemicals and gaseous hormones such as ethylene). Normal cytoplasmic analytes of plants are detected with a variety of receptors (Fujisawa et al., 2001; Friml et al., 2002) whereas normal extracellular analytes are sensed with membrane receptors (Dangl and Jones, 2001).
Sensing substances and linking the sensing to a response were recently developed in bacterial systems (Looger et al., 2003; Helling a et al., 1991). These studies and related (Swartz J R, 2001; Allert et al., 2004; US Patent Application Publications 2004/229290 and 2004/0118681 and U.S. Pat. Nos. 6,977,180 and 6,521,446) show that (1) sensor or receptor proteins can, for a substance of interest, be designed in a computer and (2) the binding of the specific substance to the computationally designed receptor can be linked to a bacterial transcriptional response system. Receptors for the bacterial system that have been designed include ones for an organophosphate surrogate of the nerve agent soman, heavy metals such as Zn2+, explosives such as TNT, herbicides such as glyphosate, and environmental pollutants such as MTBE.
There is a need in the art for monitoring systems characterized by fast feedback, ability to reset, capacity for remote evaluation, low cost to allow widespread use, ease of public recognition and the ability for operation and assessment without technically sophisticated operators or equipment.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.